2547 The flow cytometric detection of minimal residual disease (MRD) relies on the differential expression of antigens between normal and leukemic cell populations of similar lineage. In T-cell lymphoblastic leukemia (T-ALL), the principal normal populations from which leukemic blasts must be distinguished are mature T cells and NK cells, as immature T cells are not normally present in peripheral blood or bone marrow. Current methods rely on a relatively small number of antigens, some of which are not stable following therapy. In particular, immature antigens such as TdT and CD99 that distinguish leukemic and mature cells at diagnosis often revert to mature levels after therapy. The subset of T-ALL cases having expression of surface CD3 can be particularly problematic. Consequently, the identification of novel antigens to supplement those currently in use is highly desirable. We undertook a systematic search for novel antigens capable of distinguishing T-ALL from mature T cell and NK cell populations using a high-throughput flow cytometric screening technique. LyoPlates (Becton-Dickinson) containing antibodies against 242 unique antigenic specificities in a 96 well plate format were used to assay mononuclear cells obtained from 3 normal peripheral blood donors and 9 pediatric patients with T-ALL. The T-ALL cases covered the range of immunophenotypes seen in this disorder. The 9-color assay (1 detection and 8 gating fluorochromes) was performed on an LSRII and was capable of identifying discrete T cell, NK cell and Blast cell subpopulations. Comparison of the median fluorescence intensity of each of the 242 unique antigens identified CD48 as one of the few antigens that showed consistent differential expression between mature T cells and NK cells in comparison to leukemic blasts from T-ALL. To determine whether differential expression of CD48 represents a universal characteristic of the disorder, we assayed 126 consecutive pretreatment T-ALL samples from pediatric patients enrolled on Children's Oncology Group protocol AALL0434. An 8 color flow cytometric assay was employed using the following antibody combination: CD48 FITC, CD5 PE, CD16+CD56 PE-Cy5, CD3 PE-Cy7, CD8 V450 or BV421, CD4 A594, CD7 APC, CD45 APC-Cy7. Comparison of the median fluorescence intensity of CD48 between leukemic blasts and normal T and NK cells within the same specimen revealed consistently decreased expression of CD48 on leukemic blasts (see Figure 1) with some variation in intensity. This finding was highly statistically significant (p<4E-105). The stability of CD48 expression following therapy was determined by assay of CD48 expression in 126 bone marrow samples obtained at day 29 following induction chemotherapy, representing paired samples from the patients whose pretreatment samples were assayed above. The same 8 color reagent combination was used in conjunction with the standard MRD assayed utilized for the treatment protocol, a two-tube 9 color assay. Of these samples, 50 (39.7%) showed detectable MRD using the standard assay. At end induction, the ratio of CD48 median fluorescence intensity between the leukemic MRD population and normal T and NK cells within the same sample remained abnormal in most cases. It was unchanged in 26% of cases, decreased (i.e. became more aberrant) in 24%, and increased in 50%, but in most of the latter cases remained significantly <1. However, in 5 cases (10%), CD48 increased to the level seen on normal mature T and NK cells. The changes following therapy were due largely to changes in the expression of CD48 on leukemic blasts as the level of expression of normal T and NK cells was more stable. No association between apparent immunophenotypic maturational stage or surface CD3 expression was identified. We conclude that the expression of CD48 is consistently reduced on leukemic blasts from patients with pediatric T-ALL in comparison to normal mature T and NK cells at the time of diagnosis. Following therapy, the expression of CD48 undergoes modulation, but remains different from mature T and NK cells in 90% of patients. This suggests that CD48 is a useful addition to reagent combinations for the purpose of MRD assessment in pediatric T-ALL. Disclosures: Wood: BD Biosciences: Research Funding. Borowitz:BD Biosciences: Research Funding.
1436 Background: The distinction in antigen expression between normal T cells and T-ALL is currently limited by a narrow breadth of clinically useful antibodies. Our study aimed to enhance the diagnostic utility of flow cytometry in T-ALL diagnosis and minimal residual disease (MRD) detection by identifying novel aberrant antigen expression in T-ALL. Method: A high throughput flow cytometric screening method containing 242 antibodies (Lyoplate, Becton Dickinson) was used to evaluate 3 normal peripheral blood specimens and 9 diagnostic paediatric T-ALL cases. An LSRII (Becton Dickinson) was used for all flow acquisition, and Woodlist software for all analysis. The initial screening panel comprised a 9 colour assay (1 detection and 8 gating fluorochromes). All Lyoplate antibodies were analysed for differences in median fluorescence intensity between normal T cells and T-ALL populations. Subsequently, 9 specific novel antibodies were chosen for expanded testing in prospectively enrolled paediatric T-ALL cases on Children's Oncology Group protocols, using 3 additional tubes. A 9 color flow cytometric assay was designed with the following backbone antibody combination: CD3 PE-TR, CD16+CD56 PE-Cy5, CD5 PE-Cy7, CD7 V450, CD45 KO. Additionally, in each tube one of the following combinations was utilized: CD226 FITC, CD184 PE, CD229 APC or CD6 FITC, CD53 PE, CD200 APC or CD244 FITC, CD165 PE, CD27 APC. Median fluorescence intensites were compared between T-ALL populations and normal donor T cells, and the ratio of abnormal T-ALL: normal donor T cells calculated for each antibody. Results: A total of 59 patients with T-ALL were analyzed median age 8 (2–29) with 38 males, including 6 early thymic precursor (ETP) cases by St Jude's criteria. In total, 94 episodes were included: 59 pretreatment, and 35 post treatment cases at Day 29. In the T-ALL, CD27 expression was significantly reduced with a median blast/normal T cell ratio of 0.54 at diagnosis, and 0.52 post treatment. 48/53 (91%) patients had a reduced ratio <0.75 at diagnosis and 18/31 (58%) post treatment. Additionally CD6, CD226 and CD200 showed a mild reduction at diagnosis of 0.75, 0.81 and 0.84 respectively, with similar ratios seen with these 3 antibodies post treatment. The ETP cases showed a significant reduction in CD27 expression, median ratio 0.44, while reduced expression was also seen with CD6 (0.72), CD165 & CD184 (0.77), CD53 (0.78) and CD226 (0.80). There was no difference in the ratios calculated when using internal patient T cells versus normal donor T cell expression levels. Conclusion: We have demonstrated that expression levels of CD27 are consistently reduced in the majority of T-ALL and ETP patients at diagnosis and expression levels are stable at post treatment assessment. This marker has potential utility in clinical practice for diagnosis and MRD assessment. A spectrum of additional antibodies also demonstrated reduced expression, especially in ETP cases, however further patient recruitment and evaluation is required for more definitive evaluation of the utility of these antigens. Disclosures: No relevant conflicts of interest to declare.
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